In vitro passages to accumulate mutations in non-essential genes for identifying in vivo virulence factors Abstract Infertility due to tubal fibrosis/hydrosalpinx is a significant social and healthcare burden of sexually transmitted infection with Chlamydia trachomatis (CT). However, the pathogenic mechanisms remain unknown. So far, only a few virulence genes have been identified from Chlamydia muridarum (CM), a model pathogen for investigating the pathogenesis of CT due to the CM ability to induce hydrosalpinx in mice. Although successful transformation of Chlamydia has made genetic dissection of chlamydial virulence possible, genome-wide identification of virulence genes still relies on chemical-induced mutagenesis. Although these efforts have revealed the roles of chlamydial genes in promoting chlamydial infectivity, no hydrosalpinx-causing genes have been identified due to the difficulty in identifying isogenic clones because of high mutation frequencies and the failure of CT serovar L2 (used in most mutagenesis studies) to induce hydrosalpinx. Alternatively, in vitro passage has been successful in selecting chlamydial mutants resistant to chemicals. However, passaging based on the Pasteurian selection principle for accumulating mutations in non-essential genes in the absence of any selection pressure failed to induce any significant mutations in chlamydial genomes. We then developed a modified Pasteurian selection scheme by providing assistance to attachment alternately during passages of CM to maximize the recovery of mutants. After a total of 40 passages, we created 40 libraries, designated as G1 (after passage 1) to G40, in which non-essential gene mutations were accumulated as revealed by using a Bio-profiler software (patent application# 62/095,104) to align NGS reads generated from mixed templates against a reference genome sequence. We hypothesize that these mutated non-essential genes may code for virulence factors in vivo. As a proof of principle, we have isolated 4 isogenic clones, which led us to identify TC0237 and TC0668 as two novel virulence factors. We further hypothesize that TC0237 enhances CM pathogenicity by promoting ascending infection while TC0668 increases CM pathogenicity by activating hydrosalpinx-causing responses. We will use the already identified and to be identified clones from the available libraries to test these hypotheses in 3 specific aims: Continuing to isolate attenuated CM clones from libraries generated using a modified Pasteurian selection scheme (Aim I), using the attenuated CM clones to investigate the mechanisms of chlamydial ascension (Aim II) and to identify hydrosalpinx-causing mechanisms (Aim III). Accomplishing the proposed studies will significantly advance our understanding of chlamydial pathogenic mechanisms.